Abstract
Shell (cuticular) disease manifests in various forms and affects many crustaceans, including lobsters. Outbreaks of white leg disease (WLD) with distinct signs of pereiopod tissue whitening and death have been observed in cultured larvae (phyllosomas) of ornate spiny lobster Panulirus ornatus, eastern rock lobster Sagmariasus verreauxi, and slipper lobster Thenus australiensis. This study aimed to characterise and identify the causative agent of WLD through morphological and molecular (16S rRNA gene and whole genome sequencing) analysis, experimental infection of damaged/undamaged P. ornatus and T. australiensis phyllosomas, and bacterial community analysis (16S rRNA gene amplicon sequencing) of P. ornatus phyllosomas presenting with WLD during an outbreak. Bacterial communities of WLD-affected pereiopods showed low bacterial diversity and dominant abundance of Aquimarina spp. compared to healthy pereiopods, which were more diverse and enriched with Sulfitobacter spp. 16S rRNA gene Sanger sequencing of cultures from disease outbreaks identified the dominant bacterial isolate (TRL1) as a Gram-negative, long non-flagellated rod with 100% sequence identity to Aquimarina hainanensis. Aquimarina sp. TRL1 was demonstrated through comparative genome analysis (99.99% OrthoANIu) as the bacterium reisolated from experimentally infected phyllosomas presenting with typical signs of WLD. Pereiopod damage was a major predisposing factor to WLD. Histopathological examination of WLD-affected pereiopods showed masses of internalised bacteria and loss of structural integrity, suggesting that Aquimarina sp. TRL1 could enter the circulatory system and cause death by septicaemia. Aquimarina sp. TRL1 appears to have important genomic traits (e.g., tissue-degrading enzymes, gliding motility, and aggregate-promoting factors) implicated in the pathogenicity of this bacterium. We have shown that Aquimarina sp. TRL1 is the aetiological agent of WLD in cultured Palinurid and Scyllarid phyllosomas and that damaged pereiopods are a predisposing factor to WLD.
Highlights
Shell diseases of crustaceans are a global phenomenon with origins dating back more than a 100 million years (Klompmaker et al, 2016)
This study aimed to identify the causative agent of white leg disease (WLD) by (1) using 16S rRNA gene amplicon sequencing to compare bacterial communities of healthy and WLD-affected P. ornatus phyllosomas from an outbreak, (2) isolating and characterising the isolate through morphological and molecular (16S rRNA gene and whole genome sequencing) methods, and (3) fulfilling Koch’s postulates through experimental infection
Samples used for 16S rRNA gene amplicon sequencing analysis of bacterial communities were from pereiopods of three healthy and three WLD-affected P. ornatus phyllosomas removed as described and individually stored in 1 ml nucleic acid preservation solution (4 M ammonium sulphate, 25 mM sodium citrate, 10 mM EDTA, pH 5.2) at 4°C overnight followed by transfer to −20°C in preparation for further analysis
Summary
Shell (cuticular) diseases of crustaceans are a global phenomenon with origins dating back more than a 100 million years (Klompmaker et al, 2016). Shell diseases typically present as “spots” that progress to necrotic lesions of varying severity on shell surfaces. A number of behavioural and environmental drivers are hypothesised to facilitate cuticular disease, including abrasion and injury (Quinn et al, 2012), sea temperatures (Tlusty and Metzler, 2012), ocean acidification (Kunkel et al, 2012), and anthropogenic pollutants (Laufer et al, 2012). The most studied of lobster cuticular disease is ESD, which has spread along the East Coast of the United States, causing major losses in abundance, marketability, and economic value (Castro et al, 2012; Gomez-Chiarri and Cobb, 2012)
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